Roundabout4 signaling in endothelial cells

描述（由申请人提供）：在过去的四十年中，血管壁内衬的内皮细胞已成为临床关注的热点，因为易于接近允许有效的药物靶向。血管生长的失调与广泛的疾病状态相关，例如糖尿病视网膜病变、湿性年龄相关性黄斑变性和肿瘤生长。肿瘤生长依赖于血管生成。肿瘤血管生成的第一步是内皮细胞活化和内皮细胞向肿瘤的定向迁移。因此，了解定向迁移的机制对于开发可以选择性靶向肿瘤内皮细胞的治疗方法至关重要。该实验室的目标是鉴定和研究在肿瘤血管系统中选择性上调的候选基因。Robo 4是一个很好的靶点，因为它在肿瘤血管中选择性表达，并且对内皮细胞的定向迁移至关重要。此外，Robo 4的细胞表面表达允许容易地获得潜在的药物。重要的是，如果确定Robo 4上的特定信号复合物组装负责定向迁移，也可以靶向。以前，我们已经表明，robo 4是必不可少的血管生成在体内斑马鱼和激活Rho GTP酶在体外内皮细胞。初步结果表明：（A）Robo 1和Robo 4在细胞表面和细胞内水平相互作用并共享共同的信号分子;（B）包括IRSp 53、Mena、Cdc 42-GTP和Vilse在内的蛋白质复合物在内皮细胞中响应Robo 4一起组装;（C）Sry相关的高迁移率组（Sox）家族成员与Robo 4的胞质尾相互作用。我们假设：“内皮细胞表面受体Robo 1和Robo 4与Slit 2协同作用，通过细胞内信号蛋白（Cdc 42-GTP，Vilse，IRSp 53，Mena，Sox）的特定复杂组装来指导细胞迁移。“为了在目标1中验证这一假设，我们将研究Robo 1和Robo 4是否与Slit 2合作相互作用，并使用分子，生物化学和成像方法的组合在内皮细胞中直接组装Vilse和Mena的信号复合物。在目标2中，我们将确定IRSp 53，Mena，Cdc 42-GTP，Vilse和Sox与Robo 1和Robo 4的胞质尾在信号复合物中的组装机制，该信号复合物负责使用体外生物化学和体内功能测定的组合在斑马鱼中指导内皮细胞迁移。我们预计，通过实现目标1和2，我们将了解Robo 1和Robo 4如何通过解开两种分子在触发内皮细胞信号传导方面的相似性，不同信号传导分子在Robo 1和Robo 4的胞质尾上组装的顺序，它们响应的线索，以及不同信号成分的整合形成丝状伪足。每个机制步骤都是一个假定的靶点，靶向这些步骤的药物将有益于以过度血管生成和血管渗漏为特征的疾病。公共卫生相关性：肿瘤生长依赖于血管生成。我们感兴趣的是确定血管靶点，优选在肿瘤与正常内皮细胞中差异表达的细胞表面分子。该提案研究了一个这样的目标，即Robo 4。Robo 4在肿瘤血管中高度表达。肿瘤血管渗漏，血管床常呈现混乱的外观。肿瘤血管生成的第一步是内皮细胞活化和内皮细胞向肿瘤的定向迁移。因此，了解定向迁移的机制对于开发可以选择性靶向肿瘤内皮细胞的治疗方法至关重要。该建议将确定机器人在介导内皮细胞定向迁移中所利用的机制。我们提出了两个目的，调查机制所使用的机器人调解定向迁移。实现这两个目标将确定Robo 1和Robo 4如何在内皮细胞内机械组装信号复合物以响应Slit 2配体，从而协调内皮细胞的定向迁移。这项研究将确定Robo 1和Robo 4信号传导装置共同的细胞内分子，这些分子可作为药物设计的靶点，有利于与内皮细胞迁移失调相关的疾病，如与肿瘤生长相关的疾病。

DESCRIPTION (provided by applicant): Over the past four decades, endothelial cells lining the vessel wall have become an intense subject of clinical interest since ease of accessibility allows for effective drug targeting. Dysregulation of vessel growth is associated with wide range of disease states such as diabetic retinopathy, wet form of age related macular degeneration, and tumor growth. Tumor growth is angiogenesis dependent. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. The goal of this laboratory is to identify and study candidate genes that are selectively upregulated in tumor vasculature. Robo4 is an excellent target since it is expressed selectively in tumor vessels and is critical for directional migration of endothelial cells. Further, the cell surface expression of Robo4 allows easy access to potential drugs. Importantly, specific signaling complex assembly on Robo4, if identified to be responsible for directional migration can also be targeted. Previously, we have shown that robo4 is essential for angiogenesis in vivo in zebra fish and activates Rho GTPases in endothelial cells in vitro. Preliminary results suggest that: (A) Robo1 and Robo4 interact and share common signaling molecules at both the cell surface and intracellular level; (B) A complex of proteins including IRSp53, Mena, Cdc42-GTP, and Vilse together assembles in endothelial cells in response to Robo4; (C) Members of the Sry-related high-mobility-group (Sox) family interact with Robo4's cytoplasmic tail. We hypothesize that: "Endothelial cell surface receptors Robo1 and Robo4 co-operatively interact with Slit2 to direct cell migration via a specific complex assembly of intracellular signaling proteins (Cdc42-GTP, Vilse, IRSp53, Mena, Sox)." To test this hypothesis in aim 1, we will investigate whether Robo1 and Robo4 co- operatively interact with Slit2 and direct signaling complex assembly of Vilse and Mena in endothelial cells using a combination of molecular, biochemical and imaging methods. In aim 2, we will identify the mechanism of assembly of IRSp53, Mena, Cdc42-GTP, Vilse and Sox with Robo1 and Robo4's cytoplasmic tail in a signaling complex responsible for directing endothelial cell migration using a combination of in vitro biochemical and in vivo functional assays in zebra fish. We anticipate that by accomplishing aims 1 and 2, we will understand how Robo1 and Robo4 dictate the endothelial tip cell to navigate through complex milieu by unraveling the similarities between the two molecules in triggering signaling in endothelial cells, the order in which the different signaling molecules assemble on Robo1 and Robo4's cytoplasmic tail, the cues they respond to, and the integration of different signaling components to form filopodia. Each mechanistic step is a putative target and drugs targeting these steps will benefit diseases characterized by excessive angiogenesis and vascular leakage. PUBLIC HEALTH RELEVANCE: Tumor growth is angiogenesis dependent. We are interested in identifying vascular targets preferably cell surface molecules that are differentially expressed in tumor versus normal endothelial cells. This proposal studies one such target namely Robo4. Robo4 is highly expressed in tumor vessels. Tumor vessels are leaky, and the vascular bed often has a chaotic appearance. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. This proposal will identify the mechanisms utilized by Robos in mediating directional migration in endothelial cells. We propose two aims to investigate mechanisms used by Robos to mediate directional migration. Accomplishing both aims will determine how Robo1 and Robo4 mechanistically assemble a signaling complex inside endothelial cells in response to Slit2 ligand, thereby coordinating directional migration of endothelial cells. This study will identify intracellular molecules common to the Robo1 and Robo4 signaling apparatus that serve as targets for drug design benefiting conditions associated with deregulated endothelial cell migration such as those associated with tumor growth.